Multiple RNA splicing sites exist within human immunodeficiency virus type 1 (HIV-1) genomic RNA and these enable the synthesis of many mRNAs for each of several viral proteins. The effect of altered RNA processing was measured following mutagenesis of the major 5' splice donor and several cryptic, constitutive, and competing 3' splice acceptor motifs of the isolate, HIVNL4-3. Mutations that ablated constitutive splice sites led to the activation of new cryptic sites; some of these preserved function. Mutations that disrupted competing splice acceptor sites caused marked alterations in the pool of virus-derived mRNAs and, in some instances, markedly altered virus infectivity and/or the profile of virus proteins. The redundant RNA splicing signals present in the HIV-1 genome and alternatively spliced mRNAs provide a mechanism for regulating the relative proportions of HIV-1 proteins, and, in some cases, virus infectivity. A number of chimeras between the env genes of the HIVNL4-3 and HIVWMJ-2 isolates have been constructed and analyzed for function. The exchange of the first and second variable domains (V1/V2) of gp120 leads to a loss in syncytium forming ability and a partial block in gp160 processing. Virus bearing the exchanged envelope is also defective in the infection process; however, long-term passage of infected cultures resulted in the emergence of a reverted virus population. Characterization of env sequences from cells infected with revertant indicated that a single amino acid change in the major CD4 binding domain is sufficient to confer the revertant phenotype. Chimeric viruses have also been constructed involving portions of the env genes of the HIVAD8 and HIVSF162, two macrophage-tropic HIV-1 isolates. Recombinants in which regions of the envelope extending from V3 to gp41 were exchanged are defective for growth in cultures of primary human macrophages. This defect appears to be macrophage-specific, since all recombinants replicate with comparable efficiency in peripheral blood lymphocytes.